All publications and patent applications cited herein are hereby incorporated by reference into the present specification in their entireties.
Botulinum toxin-based pharmaceuticals have become a major therapeutic agent for use in non-surgical cosmetic procedures. These agents are also used extensively to treat pain and conditions associated with facial movement and head and neck disorders involving excessive muscle tone, activity or bulk.
To be economically viable, pharmaceutical preparations of Botulinum toxin must be formulated so that they have a substantial shelf-life, usually at least 6-12 months. Techniques used to maintain the shelf life of Botulinum toxin formulations have included freeze- or flash-drying the neurotoxin (e.g. BOTOX™, Botulinum toxin type A complex) or formulating the Botulinum toxin preparation as a liquid with a low pH (e.g. MYOBLOC™, Botulinum type B complex).
Pure Botulinum toxin proteins and Botulinum toxin-complexed proteins (such as, for example, Botulinum toxin protein complexed with hemaglutinin or Botulinum toxin protein complexed with non-hemaglutinin non-neurotoxin) are very stable at low, acidic pH but become increasingly unstable at higher pH (greater than about pH 6.8). Unfortunately, injectable preparations of Botulinum toxin that are formulated having an acidic pH (pH less than 7.0) cause local pain when administered to patients because of the acidic nature of the formulations. For example, MYOBLOC™, a currently-available liquid “ready-to-use” formulation of Botulinum toxin, is substantially more painful than BOTOX™. This is because the pH of MYOBLOC™ is about 5.6, which is much lower than the pH of BOTOX™, which is formulated at about pH 7.4 upon reconstitution from a freeze- or flash-dried preparation.
Elan pharmaceuticals, the makers of MYOBLOC™, have conducted studies with liquid preparations of Botulinum toxin type B in which they found that a pH of about 5.5 was necessary to achieve acceptable shelf-life stability at room temperature. Higher pHs could not be used. In contrast, BOTOX™ achieves its stability at pH 7.4 based on freeze- or flash-drying processes. The major drawback in using freeze- or flash-drying to stabilize BOTOX™ is protein denaturation which may lead to increased antigenicity. Increased antigenicity is a well-known problem associated with the production of pharmaceutical proteins that have been formulated using freeze- or flash-drying procedures. The reactions produced by increased antigenicity decrease efficacy and ultimately lead to resistance to the neurotoxin.
BOTOX™ is further hampered by the presence of sodium chloride within the pre-lyophilization fluid. The presence of salt results in additional protein denaturation due to hypertonicity which develops during the vacuum evaporation process employed in freeze- and flash-drying procedures. Furthermore, freeze- or flash-dried compositions of Botulinum toxin that began as solutions comprising a physiological saline mixture (such as is used with BOTOX™) have substantially less activity after freeze- or flash-drying than comparably prepared Botulinum toxin compositions using salt-free aqueous solutions.
To date, the effect of altering storage pH on regional denervation activity and LD50 Unit potency of injectable Botulinum toxin formulations has not been established. Although altering the pH certainly is helpful in enhancing shelf-life and stability, protein tertiary structure alteration caused by changes in pH may affect neurotoxin binding, tissue permeation and cell internalization rates.
Thus, the present invention is based on the observation that, because neither of the two Botulinum toxin formulations described above is optimal, Botulinum toxin formulations can be improved by 1) minimizing the amount of denatured protein (unnecessary antigen) in the final preparation; 2) maintaining a pH close to about 7.0 to about 7.4 in the final injection solution; and 3) increasing physician convenience.
Accordingly, the invention described herein is directed, in certain embodiments, to a novel way of storing and formulating pharmaceutically acceptable Botulinum toxin compositions so that they retain a long shelf-life, but can be administered in a form that is significantly less painful than other currently available preparations of Botulinum toxin. The inventors have found that by employing a dilution procedure immediately prior to administration to alter the Botulinum toxin formulation pH from acidic to approximately neutral or alkaline (pH=about 6.5 to about 7.4, or about 7.0 to about 7.5, preferably about 7.0 to about 7.4) a significantly less painful pharmaceutical preparation of Botulinum toxin may be made that does not sacrifice the stability or shelf-life of the preparation. The effective final potency of the preparation may be established using regional denervating assays comparing the acidic storage form of the formulation to the final diluted injectable formulation of the drug. Because the units of toxin contained in the pre-dilution liquid are labeled and known, a nomogram is available in the package insert of the preparation indicating the effect of a pH change and dilution change on the pre-injection (storage) form of the formulation.